A significant problem in organ transplantation today is the failure of current immunosuppressive strategies to significantly reduce the risk of rejection in kidney, heart, lung and pancreas transplantation more than one to two years post transplant. As a result, the tremendous gains made in the rates of one to two year survival of the transplanted organ over the last decade are largely lost at five to ten years post transplant when the majority of transplant patients, especially those receiving cadaver donor organs, have lost function in the transplanted organ.
Pathology of Allograft Rejection
There are three general stages of allograft rejection: hyperacute, acute, and chronic. In general, hyperacute rejection occurs within the first hours after transplantation. Acute rejection generally occurs in the first six to twelve months after transplantation and chronic rejection generally occurs later, usually more than one to two years post transplant.
Each stage of allograft rejection has a characteristic histopathology. Hyperacute rejection is known to be due to antibodies in the organ recipient's blood stream that react with the new organ. Hyperacute rejection results in organ failure almost immediately after transplantation.
Acute rejection is characterized by inflammation initiated by a strong T-cell based immune response to alloantigens. This T-cell based immune response can occur either directly, by cross reaction with allogeneic major histocompatibility complex (MHC) molecules, or indirectly, by the more usual route of reaction with allogeneic peptide fragments bound to host MHC molecules on antigen-presenting cells or allogeneic target cells. T-cells not only initiate the immune response, but also mediate antigen-specific effector responses. In addition, T-cells secrete soluble factors to regulate the activity of other leukocytes. For example, activated T-helper cells produce interleukins, gamma interferon and leukotrienes. This cascade of immunoregulators stimulates the attack of cytotoxic T-lymphocytes on the allograft. In irreversible rejection fatal to the allograft, these cytotoxic lymphoid cells eventually give way to larger numbers of mononuclear phagocytes and thrombocytes. The end result of the binding of thrombocytes to the allograft vascular endothelial cells is reduced blood flow, microvascular thrombosis and necrosis. Hayry, P. et al. Clin Investig 70:780-90 (1992).
In contrast to the endovascular pathology of acute rejection, chronic rejection has persistent perivascular inflammation as its most prominent feature. Often, this perivascular inflammation is accompanied by relatively low levels of lymphoid activity and arteriosclerosis of the allograft. However, compared to ordinary arteriosclerosis, which is usually defined by focal and eccentric intimal thickening, the common form of allograft arteriosclerosis is concentric and generalized intimal thickening where smooth muscle cells in the vascular intima are intermingled with some inflammatory T cells and macrophages. Hayry, P. et al. Clin Investig 70:780-90 (1992). The allograft arteriosclerosis of chronic rejection affects all intramural arteries to the level of arterioles. Other common features of chronic rejection are thinning of the vascular media and focal breaks in the internal elastic lamina.
Evidence from studies of cytokine production also supports the hypothesis that chronic rejection that is characterized by perivascular inflammation is the result of a low level immune response, that in turn induces persistent minimal damage to the allograft vascular endothelium. In response to this damage to the allograft vessels, the endothelial cells secrete growth factors, such as platelet-derived growth factor, epidermal growth factor, basic fibroblast growth factor, and transforming growth factor-beta. These growth factors stimulate the proliferation of smooth muscle cells and the migration of myocytes from the media into the intima thereby forming the arteriosclerotic lesion. Häyry, P. et al. Clin Investig 70:780-90 (1992).
Pharmaceutical Treatment of Allograft Rejection
A number of immunosuppressant drugs are known and have used for the treatment of allograft rejection. These include, for example, cyclosporin, azathioprine, FK-506, methylprednisolone, deoxypergualin, rapamycin, and mycophenylate. Today, cyclosporin (CSA) forms the basis of most immunosuppressive protocols. However, CSA's effectiveness is limited by is well known toxic side effects such as nephrotoxicity and hepatotoxicity.
In order to reduce such toxic side effects, CSA is usually combined with other immunosuppressive drugs in order to reduce the dosage of CSA to non-toxic levels. For this purpose, CSA was first combined with steroids such as methylprednisolone and later in a combination with steroids and azathioprine, which became known as triple drug therapy. However, even more potent newer immunosuppressants, such as FK-506, have been associated with toxic side effects similar to those of CSA. Schmid, T. et al. Eur Surg Res 30:61-68 (1998)
A relatively new drug, the purine nucleoside analogue, 2-chlorodeoxyadenosine (2-CDA), has already been used as a cytotoxic drug for the treatment of hairy cell leukemia, and autoimmune diseases such as autoimmune hemolytic anemia and multiple sclerosis. Because of its cytotoxicity to lymphocytes and monocytes, 2-CDA also possesses immunosuppressive properties. Although 2-CDA alone has no effect on allograft rejection, it is known to act synergistically with CSA to enhance the immunosuppressive effect of CSA when used in combination for the treatment of acute rejection. Schmid, T. et al. Eur Surg Res 30:61-68 (1998).
A number of studies have shown that the use of 2-CDA in combination with CSA may improve short-term allograft survival. One study showed that 2-chlorodeoxyadenosine, in combination with CSA reduced rejection after allogeneic small bowel transplantation in rats. Here, organ recipient rats that received a transplant of small bowel were sacrificed ten days after transplantation and the graft was examined histologically. Rats treated with a combination of 2-CDA and CSA either exhibited no evidence of graft rejection or evidence of only moderate rejection characterized by mucosal and submucosal infiltration of eosinophils and occasionally lymphocytes. However, organ recipient rats treated with either CSA or 2-CDA alone showed moderate to severe rejection including lymphocyte and polymorphonuclear granulocyte infiltration of the muscular layer and subserous fat of the graft. Schmid, T. et al. Transplantation Proceedings, 26: 1614 (1994).
Similar results were obtained in a second study involving rats that had received heart transplants. Heart allografts examined at 10 days post transplant revealed either no evidence of rejection or evidence of only mild rejection. The mild rejection was characterized by at least two foci of extensive perivascular or interstitial lymphocytic infiltration without myocyte necrosis, or one large focus of infiltration, including distortion of myocytes. Organ recipient rats treated with CSA alone exhibited either the same level of mild rejection or moderate rejection, which was characterized by multiple large lymphocytic infiltrates associated with distortion of myocyte architecture and/or myocyte necrosis. Host rats treated with 2-CDA alone exhibited severe rejection, which was characterized by extensive infiltrates as in moderate rejection that includes significant numbers of granulocytes and interstitial edema. Schmid, T., et al. Eur Surg Res 30:61-68 (1998).
Another study reported that graft survival in rats could be prolonged by the administration of CSA in conjunction with 2-CDA. Nawrocki, G., et al. Transplantation Proceedings, 28:3538-39 (1996). However, all animals died by 33 days post-transplant and there was no investigation into the pathology associated with the graft rejection.
Another study reported that organ recipient rats treated with 2-CDA and CSA displayed less of the pathology associated with graft rejection than rats treated with CSA alone at ninety days post transplant. Cramer, D. V. et al. Transplantation Proceedings, 29:616 (1997). Here, histopathological findings, including vascular intimal proliferation, perivascular fibrosis, myocardium inflammation, and myocardium fibrosis, were scored. Allograft recipients treated with CSA in combination with 2-CDA showed a reduction in the incidence and severity of vascular intimal proliferation compared to animals receiving no treatment or animals treated with CSA alone.
Although all of these studies suggest the administration of 2-CDA with CSA may limit acute allograft rejection and thus be beneficial in the improvement of short term graft survival rates, none of the studies have disclosed or suggested efficacious treatment of chronic allograft rejection.
Accordingly, what is needed is an improved method of preventing or ameliorating chronic allograft rejection, including a method of reducing the associated arteriosclerosis in human and animal allograft transplant recipients. Improved pharmaceutical compositions suitable for preventing or reducing chronic allograft rejection allograft recipients are also needed.